Many biocompatible polymeric materials have been investigated as potential plasma expanders and/or blood substitutes. Historically, there have been two approaches: 1) the use of synthetic compounds that are biocompatible, and 2) the use of biological materials that are polymeric. In the first category, materials such as hydroxyethyl starch and perfluorocarbon liquid have been evaluated (See, e.g., S. Kasper et al., J. Clin. Anesth. 13, 486-90 (2001); T. Kaneki et al., Resuscitation 52, 101-08 (2002); R. Spence et al., Art Cells, Blood Subst., Immobil. Biotech. 22, 955-63 (1999)). In the second group is gelatin, albumin, and crosslinked hemoglobin (I. Tigchelaar et al., Eur. J. Cardo-thor. Surg. 11, 626-32 (1997); S. Gould et al., World J. Surg. 20, 1200-7 (1996). More recently, alpha-keratose has been suggested. See A. Widra, U.S. Pat. No. 6,746,836 (Jun. 8, 2004).
Because the functional consequences of changing the flow properties of blood are not readily predictable, the development of plasma expanders and/or blood substitutes is a complicated matter. In arterial blood vessels (diameter>100 micron) blood viscosity is proportional to hematocrit (Hct) squared, and in the smaller vessels it is linearly proportional to Hct. In the systemic circulation, Hct is approximately constant down to 100 micron diameter vessels. It falls monotonically down to the capillaries where it is approximately half of the systemic value. The reverse occurs in the venous circulation, where it is higher than arterial because of fluid filtration in the microcirculation.
In acute conditions such as accompanying severe trauma, the decrease of Hct is not deemed dangerous until the transfusion trigger (blood hemoglobin content beyond which a blood transfusion is indicated) is reached. However, this exposes the vasculature to low blood viscosity when conventional plasma expanders are used to maintain blood volume. There appears to be no well-defined benefit to lowering blood viscosity, excepting when it is pathologically high, and lowering blood viscosity through hemodilution is considered to have no adverse effects. Richardson and Guyton determined that changes in blood viscosity are accompanied by compensatory changes in cardiac output, which compensate for changes in intrinsic oxygen carrying capacity of blood due to changes in Hct (T. Richardson et al., Am. J. Physiol. 197, 1167-70 (1959)). This was confirmed systemically and in the microcirculation (K. Messmer, Surg. Clins. N. Am. 55, 659-78 (1975); S. Mirhashemi et al., Am. J. Physiol 254 (Heart Circ. Physiol. 13) H411-16 (1988); A. Tsai et al., Int. J. Microcirc: Clin. Exp 10, 317-34 (1991)). Empirically, the transfusion trigger is set at 7 g Hb/dl (Hct˜22%).
Microvascular Hcts are lower than systemic due to the presence of a plasma layer that proportionally occupies a greater portion of the vessel lumen, thus blood viscosity is also lower. The transition from macro to microcirculation in terms of vessel dimensions, Hct, and hemodynamics is gradual. Blood rheological properties also change gradually and blood viscosity in the circulation depends on location. The reduction of Hct with a crystalloid or colloidal plasma expander tends to equalize the rheological properties of blood and viscosity throughout the circulation.
When a plasma expander is used to remedy hemorrhage, systemic Hct decreases, significantly reducing blood viscosity in large vessels due to the squared dependence of viscosity on Hct. Viscosity of blood in small vessels is much less affected since Hct is low to begin with. Conversely, small vessel blood viscosity is greatly influenced by the viscosity of the plasma expander. If its viscosity is low, blood viscosity drops significantly in the small vessels as well as in the large vessels, although for somewhat different reasons. In conventional theory, this reduction in viscosity increases blood flow and may improve oxygen delivery.
However, the literature supports the concept that high viscosity plasma is either beneficial, or has no adverse effect in conditions of extreme hemodilution. Waschke et al. found that cerebral perfusion is not changed when blood is replaced with fluids of the same intrinsic oxygen carrying capacity over a range of viscosities varying from 1.4 cp to 7.7 cp (K. Waschke et al., J. Cereb. Blood Flow & Metab. 14, 871-976 (1994)) Krieter et al., varied the viscosity of plasma by adding dextran 500 k Daltons (Da) and found that medians in tissue pO2 in skeletal muscle where maximal at a plasma viscosity of 3 cp, while for liver the maximum occurred at 2 cp (H. Krieter et al., Acta Anaest. Scad. 39, 326-44 (1995)). In general they found that up to a 3 fold increase in blood plasma viscosity had no effect on tissue oxygenation and organ perfusion when blood was hemodiluted. de Witt et al., found elevation of plasma viscosity causes sustained NO-mediated dilatation in the hamster muscle microcirculation (C. deWitt et al., Pflugers Arch. 434, 354-61 (1997)).
Hct reductions should improve blood perfusion through the increase of blood fluidity. However at a Hct near to and beyond the transfusion trigger the heart cannot further increase flow and as viscosity falls, so does blood pressure. The fall of pressure is deleterious for tissue perfusion because it decreases functional capillary density (FCD) in the normal circulation and in hypotension following hemorrhage (L. Lindbom et al., Int. J. Microcirc: Clin. Exp 4, 121-7 (1985)). FCD is a critical microvascular parameter in survival during acute blood losses. In a hamster model subjected to 4-hr 40 mmHg hemorrhagic shock, the fall of FCD accurately predicts outcome and separates survivors from non survivors when this parameter decreases below 40% of control (H. Kerger et al., Am. J. Physiol 270 (Heart. Circ. Physiol. 39), H827-36 (1996)).
High viscosity plasma restores mean arterial pressure (MAP) in hypotension without vasoconstriction. Furthermore, the shift of pressure and pressure gradients from the systemic to the peripheral circulation increases blood flow, which in combination with increased plasma viscosity maintains shear stress in the microcirculation. This is needed for shear stress dependant NO and prostaglandin release from the endothelium and to maintain FCD (J. Frangos et al., Science 227, 1477-79 (1985)). Conversely, reduced blood viscosity decreases shear stress and the release of vasodilators, causing vasoconstriction and offsetting any benefit of reducing the rheological component of vascular resistance. Since resistance depends on the 4th power of vascular radius and the 1st power of blood viscosity, the effect of reducing blood viscosity with a low viscosity plasma expander is that it reduces oxygen delivery to the tissue once blood viscosity falls below a threshold value. This threshold has been determined in our experimental model as about 2.5 cp.
Tissue perfusion with reduced blood viscosity may be deleterious at the cellular/endothelial level. There is evidence that genes are activated following changes in the mechanical environment of cells. It is also been established that the endothelium uniquely responds to changes in its mechanical and oxygen environment according to programmed genetic schemes. Among these responses is the mechanism for apoptosis (cell self destruction), which is activated through a genetically controlled suicide process that eliminates cells no longer needed or excessively damaged. In this context, hemodilution with low viscosity plasma expanders may cause cellular and tissue damage due to hypoxia and/or to the reduced vessel wall shear stress. Hypoxia/ischemia may contribute to endothelial impairment due to inflammatory reactions. Activation of endothelium, platelets and neutrophils, leading to additional damage through the liberation of cytokines, can induce endothelial apoptosis (B. Robaye et al., Am. J. Pathol. 38, 447-53 (1991)).
Studies in a hamster model show that extreme hemodilution (where Hct is 20% of control) with dextran 70 kDa, causes hypotension and a drop in FCD to near pathological values (A. Tsai et al., Proc. Natl. Acad. Sci. USA 95, 6590-5 (1998); A. Tsai. Transfusion 41, 1290-8 (2001)). This is prevented by increasing plasma viscosity so that the diluted blood has a systemic viscosity of about 2.8 cp, which was achieved by infusing dextran 500 kDa. Thus, high viscosity plasma substitutes can be an alternative to the use of blood for maintaining MAP and an adequate level of FCD (A. Tsai et al., Biorheology 38, 229-37 (2001)). However, the known high viscosity plasma expanders such as gelatin, albumin, hydroxyethyl starch, polyvinyl-pyrolidine, and dextran are all either non-human derived or synthetic. As such, each suffers from considerable limitations in their clinical applicability due to biocompatibility, cost, or both. What is needed is a fluid based on a substantially biocompatible material that is inexpensive, pathogen free and ambient storable. Resuscitation fluids based on keratins offer this potential.
Human hair is one of the few autologous tissues that can be obtained without additional surgery. It is also a rich source of keratins. Equally important, the biocompatibility of keratins within a species, and indeed across species is high, making allogenous and xenogenous keratins viable candidates for medical applications. The keratins found in hair, wool, and other keratinous tissues can be extracted and purified using methods known in the art, and used for formulating plasma substitutes with fluid properties that will maintain MAP and FCD. Depending on the species from whence the keratins come, the biocompatibility can also be optimized with human hair keratins being the most optimal. Keratin fluids are inexpensive to produce, can be sterilized, and are stable under ambient temperature storage.
However, the keratin-based fluid described in A. Widra would not appear to be the most optimized resuscitation medium based on the new paradigm of preserving FCD for three important reasons. First, the type of keratin used in the experiments was a highly hydrolyzed form of keratose, represented in Scheme 1 below, which is not likely to be capable of attaining the viscosic properties required by the application.
Second, hydrolyzed forms of keratose are compatible with blood in that they do not instigate appreciable levels of red blood cell aggregation, but their oncotic pressure is too low to be of benefit. Third, less hydrolyzed, high molecular weight forms of keratose tend to aggregate red blood cells, thus making the material deleterious to the restoration of FCD. Hence their remains a need for new approaches to developing keratin-based high viscosity plasma substitutes.